In the obligately fermentative bacterium Zymomonas mobilis, the regeneration of NAD.sup.+ depends upon two enzymes, i.e., pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1)(27, 37) Z. mobilis is the only known obligately fermentative procaryote which utilizes an Entner-Doudoroff pathway for glycolysis and, like Saccharomyces cerevisiae, produces ethanol and carbon dioxide as dominant fermentation products. We recently reported the sequence of the pyruvate decarboxylase gene from Z mobilis (9) and have now cloned and sequenced the principal alcohol dehydrogenase isozyme from this organism, the adhB gene.
Z. mobilis contains two isozymes for alcohol dehydrogenase which are readily distinguished on acrylamide gel zymograms, by ion-exchange chromatography (18, 39), and by their ability to oxidize butanol (20, 28). Although alcohol dehydrogenase II is the most abundant isozyme, both appear to contribute during fermentation (20, 28). Both isozymes have been purified, and the N-terminal amino acid sequences have been determined by polypeptide sequencing (28). The alcohol dehydrogenase I isozyme from Z. mobilis is a tetramer containing zinc with a monomeric molecular weight of between 34,000 and 40,000. The alcohol dehydrogenase II isozyme from Z. mobilis is reported to be somewhat smaller, a tetramer with a monomeric molecular weight between 31,000 and 38,000 (18, 20, 28, 36, 39). This isozyme is quite unusual, containing iron rather than zinc in its active site (28, 36). The alcohol dehydrogenase II isozyme is the most abundant alcohol dehydrogenase activity in Z. mobilis and has a high degree of specificity for ethanol as a substrate (20, 28, 39).
In this paper, we describe a novel indicator plate method for the direct detection of clones which express alcohol dehydrogenase. This technique has been used to clone the gene encoding alcohol dehydrogenase II (adhB) from Z. mobilis. This gene was sequenced, and the sites for transcriptional initiation were determined.
The invention of the subject application was disclosed in an article published in the Journal of Bacteriology, June, 1987, pp. 2591-2597.